Year : 2016 | Volume
: 1 | Issue : 3 | Page : 83--87
Advances in artificial nucleus pulposus material
Fei Yang, Jianning Zhao, Haidong Xu
Department of Orthopedics, School of Medicine, Jinling Hospital, Nanjing University, Nanjing, Jiangsu 210002
Department of Orthopedics, School of Medicine, Jinling Hospital, Nanjing University, Nanjing, Jiangsu 210002
Degenerative disc disease is very common in clinical practice, and 40% of low back pain is caused by lumbar disc degeneration. Clinical surgery can relieve symptoms in short term, but cannot achieve the purpose of cure. In discectomy surgery, to maintain a sufficient height of disc, the artificial nucleus pulposus materials such as silicone rubber, stainless steel, and hydrogel material are used to fill the defect of nucleus pulposus. With the continuous development of tissue engineering and materials science, it is possible to use stem cells or multipotent cells to cure disc disease or reverse the degenerative process and reshape the physiological function of the intervertebral disc. Now, the problem that still has to be faced in tissue engineering is how to find an extracellular scaffold which has similar structure and function of the nucleus pulposus to provide a suitable environment for the growth and physiological activity of seed cells. The article reviews the history of the development of artificial nucleus material and the current research situation.
|How to cite this article:|
Yang F, Zhao J, Xu H. Advances in artificial nucleus pulposus material.Transl Surg 2016;1:83-87
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Yang F, Zhao J, Xu H. Advances in artificial nucleus pulposus material. Transl Surg [serial online] 2016 [cited 2020 Jul 4 ];1:83-87
Available from: http://www.translsurg.com/text.asp?2016/1/3/83/191503
In the United States, totally 75%-85% persons having lower back pain (LBP) experience in their lives. Low back pain severely affects daily work and activities of the patients, and it is the most common cause of work and activity limitation, especially in young people under the age of 45.  Moreover, 40% of low back pain is caused by degenerative disc disease. The incentives of lumbar disc degeneration currently are considered to be the combined effects of the pressure, stress, nucleus pulposus cell apoptosis, and extracellular matrix metabolism problems.  Normal disc tissue is composed of nucleus pulposus in the center and annulus fibrosus in the periphery. Nucleus pulposus is mainly composed of chondrocyte-like cells and type II collagen. It has a hydrophilic gel-like structure and is proteoglycans rich, resists axial compression of the spine, and transmits the pressure to the annulus through expansion. Peripheral annulus fibrosus is composed of multilayer rows of type I collagen fibers which are gradually replaced by relatively messy type II collagen fibers inwardly. This arrangement makes the annulus have a strong tensile capacity. Under normal circumstances, the tensile strength of annulus fibrosus and the expansion pressure of the nucleus pulposus are in a relatively balanced state, to ensure a constant height of the intervertebral disc and the coordination of spine movement.  In the process of disc degeneration, the normal structure of the nucleus pulposus and annulus fibrosus is destroyed. Annulus cracking and nucleus pulposus bulging appear, followed by decreasing disc height, instability of the spine, and low back pain.
Clinically, spinal fusion is used to relieve LBP, but spinal fusion not only severely limits the movement of the spine but also leads to the segments degeneration adjacent to surgical site. Nonsurgical treatment can also relieve symptoms, but it is powerless to prevent the development of disc degeneration.  Discectomy alone has a good therapeutic effect in the short term; however, it has a high recurrence rate in the long term.  To ensure that the disc has a certain height, after discectomy surgery, artificial nucleus pulposus materials such as silicone rubber, stainless steel, and hydrogel materials are used to replace or fill the defective nucleus pulposus, and it has become a research direction and gradually developed into using tissue engineering materials to make special implants which have the similar morphology and function of human intervertebral disc structure. The latest research shows the further modified tissue engineering materials which are nanometered, functional, special structured, and looking forward to achieve the same mechanical and physical properties of normal disc. Moreover, this article summarizes the advances in artificial nucleus pulposus materials.
A computer-based retrieval was performed to search articles which describe artificial nucleus pulposus materials published between January 1, 1999, and January 1, 2016, in PubMed database with the key words "nucleus pulposus, materials" in English, and 241 articles were found in this way. The inclusion criteria are that articles are relatively new and study on artificial nucleus pulposus materials and scaffold components, structure, and construction methods. The exclusion criteria are that articles are old and repetitive and have nothing to do with standards. After filtering these articles by the inclusion and exclusion criteria, finally, 36 articles are included in this review.
Early Artificial Nucleus Pulposus Materials
It is a good idea that using artificial nucleus pulposus materials to replace the degenerated nucleus pulposus. Nachemson, a Swedish doctor, tried to inject silicone rubber into degenerated disc nucleus. Then, longitudinal pressure experiments supported the feasibility of silicone rubber as the artificial nucleus, but the lower biomechanical properties of silicone rubber limited its applications. Fernstrom tried to implant stainless steel ball into intervertebral space after intervertebral disc excision, to maintain disc height and spinal column activity. However, stainless steel ball had a phenomenon of shifting and moving into the vertebral body. Moreover, it was unable to maintain normal intervertebral height. Because of these drawbacks, it was eventually eliminated.  Hydrogels have an expansion and compression characteristics which are most similar to the nucleus pulposus in the mechanical characteristics. Bao and Yuan  found that nucleus pulposus prosthesis made by hydrogel material was able to maintain normal disc height and recover the anatomy and function of disc both in biomechanics experiments and in animal experiments. Then, Raymedia company produced semi-fluid hydrogel nucleus by polyacrylonitrile-polyacrylamide copolymers and used polyethylene fibers to wrap prosthetic disc nucleus (PDN) for clinical application. Such artificial nucleus pulposus owned a good performance in the clinical use because of its better biomechanical characteristics and stable mechanical properties. It greatly protected the spinal motor function and significantly alleviated patients' clinical symptoms in the short term. Selviaridis et al. reported a study on 36 patients with an average 100.6 months follow-up who underwent implantation of PDN. He found that although the spinal column activity of the treated patients was limited compared to normal, it was still maintained largely. There was a significant increase in disc height of these patients after the surgery. In the study, he used Oswestry Disability Index and visual analog scale to make evaluation and he found that there was a significant relief of clinical symptoms of all these patients. However, its performance was not satisfactory in the long-term efficacy. One 526 months follow-up study of 34 patients after the PDN transplantation found that 25 patients had prosthesis movement. All patients experienced different degree of low back pain, disc height decreasing, and a high proportion of endplates rupture.  Hence, this type of prosthesis cannot meet the needs of long-term replacement of human nucleus. To achieve more satisfactory long-term efficacy, it is necessary to delay or reverse pathology of intervertebral disc degeneration in the cellular level and using tissue engineering to repair and reconstruct disc has also become new options.
Nucleus Pulposus Tissue Engineering Reconstruction
Nucleus pulposus tissue engineering includes three aspects: the seed cells, scaffold, and signaling factors. Usually, for the seed cells, we can choose nucleus pulposus chondrocyte-like cells, nasal cartilage cells (NCs), bone marrow mesenchymal stem cells (MSCs), and adipose-derived MSCs (ADMSCs). Their common features are listed as below: (1) having a certain differentiation potential or specific phenotype; (2) being not likely to elicit an immune rejection; (3) having a wealth of sources and good in vitro amplification efficiency. Under certain conditions, seed cells in vitro culture can survive and proliferate, and finally they can cause the formation of the cartilage organization. The key determinant of seed cells survival and proliferation efficiency is cellular scaffolds. Scaffold plays the role of the extracellular matrix, and an ideal scaffold must meet the following requirements: (1) excellent biocompatibility and appropriate biodegradability; (2) three-dimensional porous network structure; (3) certain mechanical strength and properties; (4) simulating similar environments where cells survive in vivo.  Common hydrogel scaffold materials include natural materials, synthetic materials, and composite materials.
Natural materials are closer to the extracellular matrix in the composition such as alginates, chitosan, agarose, collagen, and chondroitin sulfate. These materials have better biological activity and biocompatibility, and they can induce the proliferation and function of seed cells. However, their poor mechanical properties cannot meet the mechanical requirements of human nucleus pulposus. Synthetic materials, such as polylactic acid, polyglycolic acid, and calcium polyphosphate, have the advantage of better mechanical properties and modified structure which is highly adjustable but have the disadvantage of poor hydrophilic and swelling capacity, no biological activity, poor biocompatibility that may have some toxic effects on cells. Hence, different natural materials and synthetic materials are combined to make composite materials which have both biocompatibility and mechanical properties. Composite materials can also be further modified and have special structure by nanotechnology. Current studies about different kinds of hydrogel scaffolds with their special treatment and modification methods will be mentioned below.
Type II collagen-hyaluronic acid composites
Type II collagen is an important component of the extracellular matrix of the nucleus pulposus, with good biocompatibility. During the nucleus pulposus tissue degeneration process, the type II collagen content decreases significantly. Type II collagen-based hydrogels compared to type I collagen-based hydrogels can significantly induce adipose-derived stem cells differentiating into cartilage direction, promoting generation of cartilage.  To improve the mechanical properties and the resistance to enzymatic degradation of type II collagen-based hydrogels, both glutaraldehyde and carbodiimide were used as the cross-linking agent. However, the toxic effects on the seed cells were observed in experiments. , There is a higher level of hyaluronic acid in normal human nucleus pulposus, which can promote the production of nucleus pulposus extracellular matrix, nucleus pulposus cells proliferation, and cartilage phenotype maintenance.  Roman made a special collagen-low molecular weight hyaluronic acid semi-interpenetrating network structure which was added by gelatin particles wrapped with transforming growth factor-β3 factor. He found that the viscoelasticity of this material was excellent, and MSCs could grow along the collagen fibers with high cell activity and cartilage differentiation.  Halloran et al. used type II collagen-hyaluronic acid scaffold as a carrier to culture nucleus pulposus cells and found that the nucleus pulposus cell phenotype had better maintenance. Then Collin et al. used poly (ethylene glycol) ether tetrasuccinimidyl glutarate (4S-StarPEG) as a cross-linking agent to make the type II collagen crosslink to form a hydrogel and hyaluronic acid was mixed into hydrogel in different proportions. Cell experiments in vitro found that 4S-StarPEG had low cytotoxicity, fast gelation speed, and high controllability. This hydrogel nucleus could promote cell survival and had nothing to do with the ratio of added hyaluronic acid. Although type II collagen-hyaluronic acid hydrogel has good biocompatibility, it has lower mechanical properties compared to synthetic materials such as polylactic acid. It cannot meet the requirement of stress which exist in normal intervertebral disc.
In conclusion, type II collagen-hyaluronic acid hydrogel is a good choice for artificial nucleus pulposus implantation because it is the most similar to the normal extracellular matrix of nucleus pulposus.  However, it should be further modified to get better mechanical properties for clinical applications.
Alginate is a natural polysaccharide present in the brown algae, and it can polymerize to form a hydrogel under the role of cross-linking agents which can be easily separated such as divalent cations. This hydrogel has a network structure, and it meets the environmental requirements for cell survival. Alginate has high biocompatibility and low cytotoxicity, while the cost is relatively low. Hence, earlier it has been used in the preparation of injectable hydrogels.  However, the polymerization process of alginate is not uniform, and the surface of the solution once polymerizes first that will limit further polymerization of lower solution and affect its mechanical strength after gelation.  Thereby, controlling alginate gelation process is essential for preparing such hydrogels. Most studies use calcium chloride (CaCl 2 ) as a cross-linking agent, but the calcium ion has a high degree of dissociation and the cross-linking process is not under control, and hydrogels have different characters after gelling. Growney et al.  used calcium carbonate (CaCO 3 ) as a cross-linking agent because the dissociation degree of CaCO 3 can be controlled by glucono-δ-lactone (GDL). He adjusted GDL hydrolytic degree by controlling the temperature and pH value to regulate the dissociation degree of CaCO 3 , so the cross-linking process of alginate becomes controllable. Slow gelling alginate hydrogels prepared in this way have been greatly improved on the mechanical properties, viscoelasticity, diffusion, water retention, and swelling ratio. In summary, as a type of natural material, alginate has high biocompatibility but the low mechanical properties limit its application. However, cross-linking process has become controllable with the development of the technology, and the mechanical properties of alginate hydrogels have been improved. Hence alginate becomes a possible choice for nucleus pulposus tissue engineering again.
Chitosan is a polysaccharide consisting of N-acetylglucosamine units and glucosamine units randomly distributed, and it is similar in structure and function with glycosaminoglycans (GAGs). Chitosan can form hydrogels through self-association or covalently crosslinking.  Chitosan hydrogels can promote the growth and differentiation of chondrocytes and induce cartilage-specific extracellular matrix generating.  While chitosan hydrogels have a natural cationic character which can combine to anion binding proteoglycans produced by cartilage cells. Manitha used chitosan and polyhydroxybutyrate-co-valerate (PHBV) to make composite hydrogels with nanoparticles of chondroitin sulfate added and the preparation process did not need cross-linking agent. The hydrogels have similar viscoelasticity to the normal nucleus pulposus that can withstand the stress equal to lumbar spine daily activities such as lying down (0.01 MPa), sitting (0.5 MPa), and standing (1.0 MPa). The experiment in vitro on seed cells found that compared to chitosan PHBV hydrogels without chondroitin sulfate nanoparticles, hydrogels added with chondroitin sulfate nanoparticles significantly enhanced the activity and capacity of rat ADMSCs differentiating into cartilage.  Actually, chitosan has been widely used in tissue engineering because of its biological properties.  As a scaffold material, chitosan is cheap and easy to get, and it can be made into an injectable hydrogel for further application.
Gellan gum hydrogels
Gellan gum (GG) is a kind of extracellular polysaccharide produced by Sphingomonas elodea. It is generated by glucose-glucuronide-glucose-rhamnose as a repeating unit and it has a gel-like nature. Oliveira et al. used it in tissue engineering to repair cartilage and achieved good results. Silva-Correia et al. used the low acyl GG with methacrylic acid to form methacrylated GG (GG-MA) copolymer structure. Compared to the unmodified GG, it had better mechanical performance and low degradation rate. Such hydrogels can gel by ionic crosslinking or photo-crosslinking manner, and in human bone marrow MSCs cultured experiments, the cells can grow and survive up to 21 days.  Thereafter, Roman experiments in vitro found that the 2 different crosslinking (GG-MA) both will not induce in vitro cytotoxicity and pro-inflammatory, but ionic crosslinking GG-MA has a better biological compatibility, and after subcutaneous implantation, it can maintain cartilage formation potential of bone marrow MSCs and NCs.  GG owns good biological compatibility and appropriate biodegradability, which is very cheap. However, GG must be modified to get better mechanical property before it was used. The agent which is added to GG may have certain cytotoxicity.
Hyaluronic acid as a common GAG played a key role in maintaining the normal physiological function of nucleus pulposus tissue matrix. The long-chain of hyaluronic acid can be used as a skeleton for aggrecan, which forms a high negative charge that increases the absorption of water and ensure nucleus pulposus mechanical properties. Hyaluronic acid also can contribute to the development and promotion of the extracellular matrix of nucleus pulposus and maintains chondrocyte phenotypes of nucleus pulposus cells.  In view of the excellent biological functions of hyaluronic acid, it is often combined with other materials to form a composite hydrogel. Claire combined polyethylene glycol with different molecular weight of hyaluronic acid to form a composite hydrogel and found that with hyaluronic acid molecular weight increasing, composite hydrogel stiffness was greater. Cell culture in vitro, composite hydrogel with low molecular weight hyaluronic acid is more conducive to the survival of nucleus pulposus cells and synthesis of extracellular sulfate GAG.  Sivan et al. used monomers of sodium 2-acrylamido-2-methylpropane sulfonic acid and potassium salt of 3-sulfopropyl acrylate to make GAG analogs, which had similar biological performance of the GAGs. In summary, GAGs hydrogel has an excellent water-absorbing quality, and it can ensure the permeability of nucleus pulposus. Hence, GAGs can be combined to other materials to make the hydrogel have good mechanical performance.
Polyphosphates are a composite of phosphate repeating units, belonging to the biologically active compound, and are widely used as a preservative in toothpaste, baked goods, and meat industries.  It exists in all prokaryotic and eukaryotic cells, and among the lower organisms such as bacteria and fungi, it is an alternative source of energy. In mammalian cell systems, it is able to regulate a variety of important physiological processes.  In vitro experiments, polyphosphate hydrogels strengthen the metabolism of cartilage cells, promote the formation of cartilage.  Gawri et al.  used polyphosphate hydrogels scaffold to culture nucleus pulposus cells in vitro and found that the hydrogels played a regulatory role of the nucleus pulposus extracellular matrix production and accumulation by the polyphosphate concentration and the length of molecular chains. Hence, it has been proved that polyphosphates own certain biological compatibility. Compared to natural materials such as type II collagen, polyphosphates have higher mechanical strength, and this merit makes polyphosphates be a good choice for nucleus pulposus tissue engineering. However, the drawback of polyphosphates is that it has low degradability in vivo. The goal of tissue engineering is to induce nucleus pulposus cells to produce extracellular matrix and to reconstruct nucleus pulposus eventually, so it must be ensured that the scaffold materials are able to degraded after the extracellular matrix formation. The low degradability of polyphosphates limits its application for nucleus pulposus tissue engineering; however, it is still hopeful that polyphosphates become more biodegradable with the development of material science. In short, polyphosphates may be a possible choice for nucleus pulposus tissue engineering but not the ideal choice at present.
Because the nucleus pulposus is part of the human immune privilege tissue, so there is a high feasibility of the allograft of stem cells or function cells. Using tissue engineering to reverse or repair intervertebral disc degeneration at the cellular level is the focus of current research. The most important current problems need to be addressed in tissue engineering is to develop a extracellular scaffold having similar structure and function of the cytoskeleton, providing a favorable microenvironment for the survival and activity of seed cells, promoting seed cells synthesize extracellular matrix, owning adequate mechanical strength, and mechanical properties to maintain disc height and spine motion. With the development and progress of materials science, scaffold materials will be further modified such as nanostructure, and ultimately achieve the functional requirements of the nucleus. However, the majority of research and development of the scaffold at this stage still stay in vitro experimental level, and lack of the animal testing data. There are inherent differences between animal models and human disc degeneration that also increases uncertainty in clinical use in patients in future. Hence, there is a certain distance from the clinical use.
In conclusion, first, natural materials such as alginates and chitosan have better biological activity and biocompatibility but with poor mechanical properties compared to synthetic materials. Second, synthetic materials such as polyphosphates own excellent mechanical performance and modified structure which is highly adjustable. What's more, different natural materials and synthetic materials can be combined to make composite materials which have both biocompatibility and mechanical properties. Finally, the disc tissue engineering brings possibility to cure degenerative disc disease with the development and progress of materials science, but the road to achieve its clinical use is full of challenges.
Financial support and sponsorship
This study was supported by National Natural Science Foundation of China (Grant Number: 81501925).
Conflicts of interest
There are no conflicts of interest.
|1||Andersson GB. Epidemiological features of chronic low-back pain. Lancet 1999;354 (9178):581-5.|
|2||Molinos M, Almeida CR, Caldeira J, Cunha C, Gonçalves RM, Barbosa MA. Inflammation in intervertebral disc degeneration and regeneration. J R Soc Interface 2015;12 (104):20141191.|
|3||Kepler CK, Ponnappan RK, Tannoury CA, Risbud MV, Anderson DG. The molecular basis of intervertebral disc degeneration. Spine J 2013;13 (3):318-30.|
|4||Iatridis JC, Nicoll SB, Michalek AJ, Walter BA, Gupta MS. Role of biomechanics in intervertebral disc degeneration and regenerative therapies: What needs repairing in the disc and what are promising biomaterials for its repair? Spine J 2013;13 (3):243-62.|
|5||Xueming C, Yadong L, Songjie X, Libin C, Xu Z, Zhenshan Y, Xiuzhi S, Hua G. Result of lumbar discectomy for single-level lumbar intervertebral disc herniation: A ten-year report. Chin Spine Spinal Cord 2011;21 (8):644-9.|
|6||Sakalkale DP, Bhagia SA, Slipman CW. A historical review and current perspective on the intervertebral disc prosthesis. Pain Physician 2003;6 (2):195-8.|
|7||Bao QB, Yuan HA. Prosthetic disc replacement: The future? Clin Orthop Relat Res 2002;(394):139-45.|
|8||Selviaridis P, Foroglou N, Tsitlakidis A, Hatzisotiriou A, Magras I, Patsalas I. Long-term outcome after implantation of prosthetic disc nucleus device (PDN) in lumbar disc disease. Hippokratia 2010;14 (3):176-84.|
|9||Ma YZ, Xue HB, Chen X, Guo LX, Li HW, Liu HR. The mid- or long-term clinical results of prosthetic disc nucleus replacement in the treatment of lumbar disc disease. Zhonghua Wai Ke Za Zhi 2008;46 (5):350-3.|
|10||Yiguang B, Gang F. The development of research of tissue engineering on intervertebral disc degeneration. Med J West China 2014;26 (8):1100-2.|
|11||Lu Z, Doulabi BZ, Huang C, Bank RA, Helder MN. Collagen type II enhances chondrogenesis in adipose tissue-derived stem cells by affecting cell shape. Tissue Eng Part A 2010;16 (1):81-90.|
|12||Orban JM, Wilson LB, Kofroth JA, El-Kurdi MS, Maul TM, Vorp DA. Crosslinking of collagen gels by transglutaminase. J Biomed Mater Res A 2004;68 (4):756-62.|
|13||Saito H, Murabayashi S, Mitamura Y, Taguchi T. Characterization of alkali-treated collagen gels prepared by different crosslinkers. J Mater Sci Mater Med 2008;19 (3):1297-305.|
|14||Pek YS, Kurisawa M, Gao S, Chung JE, Ying JY. The development of a nanocrystalline apatite reinforced crosslinked hyaluronic acid-tyramine composite as an injectable bone cement. Biomaterials 2009;30 (5):822-8.|
|15||Tsaryk R, Gloria A, Russo T, Anspach L, De Santis R, Ghanaati S, Unger RE, Ambrosio L, Kirkpatrick CJ. Collagen-low molecular weight hyaluronic acid semi-interpenetrating network loaded with gelatin microspheres for cell and growth factor delivery for nucleus pulposus regeneration. Acta Biomater 2015;20:10-21.|
|16||Halloran DO, Grad S, Stoddart M, Dockery P, Alini M, Pandit AS. An injectable cross-linked scaffold for nucleus pulposus regeneration. Biomaterials 2008;29 (4):438-47.|
|17||Collin EC, Grad S, Zeugolis DI, Vinatier CS, Clouet JR, Guicheux JJ, Weiss P, Alini M, Pandit AS. An injectable vehicle for nucleus pulposus cell-based therapy. Biomaterials 2011;32 (11):2862-70.|
|18||Li CQ, Huang B, Luo G, Zhang CZ, Zhuang Y, Zhou Y. Construction of collagen II/hyaluronate/chondroitin-6-sulfate tri-copolymer scaffold for nucleus pulposus tissue engineering and preliminary analysis of its physico-chemical properties and biocompatibility. J Mater Sci Mater Med 2010;21 (2):741-51.|
|19||Drury JL, Mooney DJ. Hydrogels for tissue engineering: Scaffold design variables and applications. Biomaterials 2003;24 (24):4337-51.|
|20||Van Tomme SR, Storm G, Hennink WE. In situ gelling hydrogels for pharmaceutical and biomedical applications. Int J Pharm 2008;355 (1-2):1-18.|
|21||Growney Kalaf EA, Flores R, Bledsoe JG, Sell SA. Characterization of slow-gelling alginate hydrogels for intervertebral disc tissue-engineering applications. Mater Sci Eng C Mater Biol Appl 2016;63:198-210.|
|22||Suh JK, Matthew HW. Application of chitosan-based polysaccharide biomaterials in cartilage tissue engineering: A review. Biomaterials 2000;21 (24):2589-98.|
|23||Chicatun F, Pedraza CE, Muja N, Ghezzi CE, McKee MD, Nazhat SN. Effect of chitosan incorporation and scaffold geometry on chondrocyte function in dense collagen type I hydrogels. Tissue Eng Part A 2013;19 (23-24):2553-64.|
|24||Nair MB, Baranwal G, Vijayan P, Keyan KS, Jayakumar R. Composite hydrogel of chitosan-poly(hydroxybutyrate-co-valerate) with chondroitin sulfate nanoparticles for nucleus pulposus tissue engineering. Colloids Surf B Biointerfaces 2015;136:84-92.|
|25||Roughley P, Hoemann C, DesRosiers E, Mwale F, Antoniou J, Alini M. The potential of chitosan-based gels containing intervertebral disc cells for nucleus pulposus supplementation. Biomaterials 2006;27 (3):388-96.|
|26||Oliveira JT, Santos TC, Martins L, Picciochi R, Marques AP, Castro AG, Neves NM, Mano JF, Reis RL. Gellan gum injectable hydrogels for cartilage tissue engineering applications: In vitro studies and preliminary in vivo evaluation. Tissue Eng Part A 2010;16 (1):343-53.|
|27||Silva-Correia J, Oliveira JM, Caridade SG, Oliveira JT, Sousa RA, Mano JF, Reis RL. Gellan gum-based hydrogels for intervertebral disc tissue-engineering applications. J Tissue Eng Regen Med 2011;5 (6):e97-107.|
|28||Silva-Correia J, Gloria A, Oliveira MB, Mano JF, Oliveira JM, Ambrosio L, Reis RL. Rheological and mechanical properties of acellular and cell-laden methacrylated gellan gum hydrogels. J Biomed Mater Res A 2013;101 (12):3438-46.|
|29||Tsaryk R, Silva-Correia J, Oliveira JM, Unger RE, Landes C, Brochhausen C, Ghanaati S, Reis RL, Kirkpatrick CJ. Biological performance of cell-encapsulated methacrylated gellan gum-based hydrogels for nucleus pulposus regeneration. J Tissue Eng Regen Med 2014. DOI: 10.1002/term.1959.|
|30||Chung C, Erickson IE, Mauck RL, Burdick JA. Differential behavior of auricular and articular chondrocytes in hyaluronic acid hydrogels. Tissue Eng Part A 2008;14 (7):1121-31.|
|31||Jeong CG, Francisco AT, Niu Z, Mancino RL, Craig SL, Setton LA. Screening of hyaluronic acid-poly (ethylene glycol) composite hydrogels to support intervertebral disc cell biosynthesis using artificial neural network analysis. Acta Biomater 2014;10 (8):3421-30.|
|32||Sivan SS, Roberts S, Urban JP, Menage J, Bramhill J, Campbell D, Franklin VJ, Lydon F, Merkher Y, Maroudas A, Tighe BJ. Injectable hydrogels with high fixed charge density and swelling pressure for nucleus pulposus repair: Biomimetic glycosaminoglycan analogues. Acta Biomater 2014;10 (3):1124-33.|
|33||Gunther NW 4 th . Effects of polyphosphate additives on Campylobacter survival in processed chicken exudates. Appl Environ Microbiol 2010;76 (8):2419-24.|
|34||Rao NN, Kornberg A. Inorganic polyphosphate regulates responses of Escherichia coli to nutritional stringencies, environmental stresses and survival in the stationary phase. Prog Mol Subcell Biol 1999;23:183-95.|
|35||St-Pierre JP, Wang Q, Li SQ, Pilliar RM, Kandel RA. Inorganic polyphosphate stimulates cartilage tissue formation. Tissue Eng Part A 2012;18 (11-12):1282-92.|
|36||Gawri R, Shiba T, Pilliar R, Kandel R. Inorganic polyphosphates enhances nucleus pulposus tissue formation in vitro. J Orthop Res 2016. DOI: 10.1002/jor.23288.|